Recombinant Mesocricetus auratus Class E basic helix-loop-helix protein 22 (BHLHE22)

What is the primary function of BHLHE22 in neural development?

BHLHE22 functions as an important regulator of neurogenesis and neuronal differentiation in the central nervous system (CNS). It serves as a transcriptional repressor by binding to sequence-specific DNA elements and recruiting PRDM8, a transcription factor that inhibits DNA methylation . This repressor complex regulates target genes involved in neural development, particularly those mediating axonal guidance in dorsal telencephalic neurons and controlling inhibitory synaptic interneurons in the dorsal horn .

Mouse studies demonstrate that BHLHE22 is critically involved in corpus callosum formation, as mice lacking bhlhe22 show nearly complete loss of three brain commissures: the corpus callosum, hippocampal commissure, and anterior commissure . These findings indicate that BHLHE22 plays an essential role in establishing proper axonal connectivity between the cerebral hemispheres during development.

What is the expression pattern of BHLHE22 in mammals?

BHLHE22 expression is highly tissue-specific, being expressed exclusively in the central nervous system (CNS) and retina . As a Class II bHLH protein, this restricted expression pattern contrasts with Class I bHLH proteins that are ubiquitously expressed . Within the CNS, BHLHE22 is required for the differentiation of neurons in several domains, including:

• The dorsal horn of the spinal cord

• The dorsal cochlear nucleus in the brainstem

• Retinal amacrine cells

• The dorsal telencephalon

This restricted expression pattern reflects BHLHE22's specialized role in neuronal differentiation and circuit formation in specific brain regions.

What are the optimal methods for recombinant expression and purification of BHLHE22?

Recommended Expression System:
For functional studies of recombinant Mesocricetus auratus BHLHE22, a eukaryotic expression system is preferable due to the potential requirement for post-translational modifications. The following methodological approach is recommended:

• Expression vector selection: Use a mammalian expression vector (e.g., pCDNA3.1) for cell culture experiments or a baculovirus expression system for larger-scale protein production.

• Affinity tags: Incorporate a small affinity tag (His6 or FLAG) at either the N- or C-terminus, avoiding the HLH domain to prevent interference with dimerization.

• Purification protocol:

  • Initial capture using affinity chromatography (Ni-NTA for His-tagged proteins)

  • Intermediate purification using ion-exchange chromatography

  • Final polishing using size-exclusion chromatography to separate monomers from dimers and aggregates

• Buffer optimization: Include reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues and glycerol (10-15%) to enhance stability.

• Activity verification: Confirm DNA-binding activity using electrophoretic mobility shift assays (EMSAs) with E-box containing oligonucleotides.

How can CRISPR-Cas9 genome editing be used to study BHLHE22 function?

CRISPR-Cas9 genome editing provides powerful approaches for investigating BHLHE22 function in neural development. Key methodological considerations include:

• Knockout studies:

  • Design sgRNAs targeting the single exon of BHLHE22, preferably in the 5' region to ensure complete loss of function

  • For hamster models, use embryonic fibroblasts or validated hamster cell lines

  • Validate knockout efficiency using sequencing, RT-PCR, and Western blotting

• Knock-in approaches:

  • For studying disease-associated variants, introduce specific mutations such as those affecting the HLH domain (e.g., p.Glu251Gln, p.Met255Arg, p.Leu262Pro) that have been identified in human patients

  • For protein interaction studies, create fluorescent fusion proteins by inserting tags at non-critical regions

• Domain analysis:

  • Generate domain-specific deletions to assess the functional importance of individual protein domains

  • Focus on the highly conserved HLH domain (amino acids ~240-300) that mediates dimerization and DNA binding

• Temporal control:

  • Implement inducible CRISPR systems (e.g., Tet-On/Off) to study BHLHE22 function at specific developmental stages

• Validation and phenotyping:

  • Assess neural development using neurite outgrowth assays, cell migration studies, and differentiation markers

  • Analyze morphological changes in corpus callosum formation using histological and imaging techniques

What experimental approaches are effective for identifying BHLHE22 target genes?

Identifying direct target genes of BHLHE22 is crucial for understanding its role in neural development. The following methodological approaches are recommended:

• Chromatin Immunoprecipitation sequencing (ChIP-seq):

  • Use validated anti-BHLHE22 antibodies or epitope-tagged recombinant BHLHE22

  • Look for enrichment at E-box motifs (CANNTG) in regulatory regions

  • Focus on neuronal genes, particularly those involved in axon guidance and synapse formation

  • Compare results with published datasets from mouse studies showing BHLHE22 binding sites

• RNA-sequencing after BHLHE22 manipulation:

  • Perform differential expression analysis following BHLHE22 overexpression or knockdown

  • Use neuronal cell lines or primary neuronal cultures for physiological relevance

  • Apply time-course analyses to distinguish primary from secondary transcriptional effects

• CUT&RUN or CUT&Tag assays:

  • These newer techniques offer higher resolution and lower background than traditional ChIP-seq

  • Particularly useful when working with limited cell numbers from specific neuronal populations

• Validation studies:

  • Confirm direct regulation using reporter assays with identified regulatory elements

  • Perform site-directed mutagenesis of E-box motifs to verify BHLHE22 binding specificity

  • Use EMSA assays to confirm physical interaction with predicted binding sites

A key target of BHLHE22 identified in mouse studies is Cadherin-11 (CDH11), a cell-cell adhesion protein that regulates neural circuitry assembly . This and other targets should be prioritized for validation in hamster models.

How do BHLHE22 variants impact brain development and neurological function?

Recent studies have identified both monoallelic and biallelic BHLHE22 variants associated with a neurodevelopmental disorder in humans. The pathological mechanisms and phenotypic manifestations include:

• Structural brain abnormalities:

  • Partial or complete agenesis of the corpus callosum (ACC) is observed in the majority of affected individuals

  • This correlates with findings in bhlhe22 knockout mice, which show nearly complete loss of three brain commissures, indicating an evolutionarily conserved role in axon guidance

• Clinical manifestations include:

  • Absent or limited speech

  • Severely impaired motor abilities

  • Intellectual disability (ID)

  • Involuntary movements

  • Autistic traits with stereotypies

  • Abnormal muscle tone

  • Additional features including epilepsy and eye anomalies in some cases

• Genotype-phenotype correlations:

  • Four identified de novo missense variants (p.Glu251Gln, p.Met255Arg, p.Leu262Pro) are located in the highly conserved HLH domain and follow an autosomal dominant inheritance pattern

  • A recurrent homozygous frameshift variant (p.Gly74Alafs*18) in the glycine-rich region follows an autosomal recessive inheritance pattern

  • These findings suggest different molecular mechanisms may be involved in dominant versus recessive forms of BHLHE22-associated disorders

• Molecular mechanisms:

  • Variants in the HLH domain likely disrupt protein dimerization and subsequent DNA binding

  • The frameshift variant leads to a truncated protein lacking the functional HLH domain

  • Both mechanisms result in disrupted regulation of target genes critical for neural development and axon guidance

What are the implications of BHLHE22 expression in cancer research?

Beyond its role in neurodevelopment, BHLHE22 has significant implications in cancer biology, particularly in endometrial cancer:

• Expression patterns:

  • BHLHE22 protein expression is significantly downregulated in endometrial cancer compared to normal endometrium

  • This downregulation is associated with hypermethylation of the BHLHE22 promoter

• Functional effects in cancer cells:

  • Overexpression of BHLHE22 in endometrial cancer cell lines results in:

    • Significant decrease in cell proliferation (103.6% and 217.9% decrease in HEC1A and Ishikawa cell lines, respectively)

    • Reduced cellular mobility (38.5% and 34.8% decrease in HEC1A and Ishikawa cell lines)

  • These findings suggest a potential tumor suppressor role for BHLHE22

• Prognostic associations:

  • High BHLHE22 expression is associated with:

    • Microsatellite-instable subtype

    • Endometrioid type

    • Tumor grade

    • Patient age

  • High expression correlates with favorable survival outcomes

• Immune microenvironment interactions:

  • BHLHE22 expression positively correlates with stromal, immune, and ESTIMATE scores

  • This suggests BHLHE22 may regulate host immune responses and create a "hot" immunogenic microenvironment

  • BHLHE22 could serve as a potential biomarker for predicting immune checkpoint inhibitor response

While these findings are from studies of human cancer, they suggest potential directions for investigating BHLHE22 function in comparative oncology models using hamster BHLHE22.

What are the challenges in analyzing BHLHE22 protein-protein interactions?

Investigating the interactome of BHLHE22 presents several technical challenges that researchers should consider:

• Dimerization partners:

  • As a Class II bHLH protein, BHLHE22 likely forms heterodimers with Class I bHLH proteins

  • Identifying the specific dimerization partners in different neuronal populations requires cell-type specific approaches

  • Methods to consider include BioID or APEX proximity labeling in defined neuronal populations

• Repressor complex formation:

  • BHLHE22 forms a repressor complex with PRDM8 to regulate target gene expression

  • Stoichiometry and dynamics of this complex remain to be fully characterized

  • Techniques such as blue native PAGE, analytical ultracentrifugation, or multi-angle light scattering can help determine complex composition

• Post-translational modifications:

  • Potential phosphorylation, SUMOylation, or other modifications may regulate BHLHE22 activity

  • Phosphoproteomic analysis of recombinant BHLHE22 can identify modification sites

  • Site-directed mutagenesis of identified sites can determine functional significance

• Tissue-specific interactions:

  • BHLHE22 may have different interaction partners in retina versus CNS

  • Single-cell approaches (e.g., single-cell co-immunoprecipitation) can help resolve cell-type specific interactions

• Structural considerations:

  • The N-terminal proline-rich domain and C-terminal alanine-rich region may mediate specific protein-protein interactions beyond dimerization

  • Truncation constructs can help map interaction domains

How can epigenetic regulation of BHLHE22 expression be effectively studied?

Given the evidence for epigenetic regulation of BHLHE22 in cancer contexts, the following methodological approaches are recommended for studying its epigenetic regulation:

• DNA methylation analysis:

  • Bisulfite sequencing of the BHLHE22 promoter region to quantify CpG methylation at single-nucleotide resolution

  • Methylation-specific PCR for targeted analysis of specific CpG sites

  • Comparison between different tissue types and developmental stages

• Chromatin structure and accessibility:

  • ATAC-seq to assess chromatin accessibility at the BHLHE22 locus

  • DNase-seq or MNase-seq to map nucleosome positioning

  • Hi-C or Capture-C to analyze three-dimensional chromatin interactions

• Histone modifications:

  • ChIP-seq for histone marks associated with active (H3K4me3, H3K27ac) and repressive (H3K27me3, H3K9me3) chromatin at the BHLHE22 locus

  • Sequential ChIP to identify bivalent domains potentially regulating developmental expression

• Transcription factor binding:

  • ChIP-seq for transcription factors that potentially regulate BHLHE22 expression

  • Focus on neurodevelopmental transcription factors that may activate BHLHE22 in neural lineages

• Functional validation:

  • CRISPR-dCas9 with epigenetic effectors (e.g., DNMT3A, TET1, HDAC1) to manipulate the epigenetic state of the BHLHE22 locus

  • Reporter assays with methylated versus unmethylated promoter constructs

What are the considerations for designing antibodies against hamster BHLHE22?

Developing specific antibodies against Mesocricetus auratus BHLHE22 requires careful epitope selection and validation strategies:

• Epitope selection criteria:

  • Choose regions with high antigenicity and surface accessibility

  • Avoid the highly conserved HLH domain if species-specificity is required

  • Consider the N-terminal region (amino acids 50-100) or C-terminal region (amino acids 300-350) for hamster-specific antibodies

  • For pan-species antibodies, target the highly conserved HLH domain

• Antibody types to consider:

  • Polyclonal antibodies for multiple epitope recognition

  • Monoclonal antibodies for consistency and specificity

  • Recombinant antibodies for reproducibility and reduced batch variation

• Validation methods:

  • Western blotting against recombinant hamster BHLHE22 and tissue lysates

  • Immunoprecipitation followed by mass spectrometry

  • Immunohistochemistry with peptide competition controls

  • Testing in BHLHE22 knockout models as negative controls

• Applications-specific considerations:

  • For ChIP applications, select antibodies against surface-exposed epitopes that do not interfere with DNA binding

  • For immunofluorescence, confirm accessibility of the epitope in fixed tissues

  • For co-immunoprecipitation, ensure the epitope is not involved in protein-protein interactions

What are promising approaches for studying BHLHE22 in neurological disorders?

Based on recent findings linking BHLHE22 variants to neurodevelopmental disorders, several research directions merit investigation:

• Development of animal models:

  • Generate hamster models carrying the equivalent of human disease-associated variants

  • Use CRISPR-Cas9 to introduce specific mutations in the HLH domain (e.g., p.Glu251Gln, p.Met255Arg, p.Leu262Pro)

  • Create a knockout model to study complete loss of function

• Developmental trajectory analysis:

  • Use time-course studies to determine when and how BHLHE22 dysfunction affects neural development

  • Employ lineage tracing to track the fate of neurons expressing mutant BHLHE22

  • Perform single-cell transcriptomics at different developmental stages to identify cell populations most affected

• Circuit-level investigations:

  • Apply connectomics approaches to map altered neural circuits in animal models

  • Use calcium imaging to assess functional connectivity

  • Employ optogenetics to manipulate specific neuronal populations affected by BHLHE22 dysfunction

• Therapeutic explorations:

  • Test whether reintroduction of functional BHLHE22 can rescue developmental defects if administered during critical periods

  • Investigate whether targeting downstream effectors can compensate for BHLHE22 dysfunction

  • Explore potential gene therapy approaches for early intervention

• Translational biomarkers:

  • Develop imaging biomarkers to detect subtle corpus callosum abnormalities

  • Identify molecular signatures in accessible tissues that correlate with BHLHE22 function in the brain

  • Establish preclinical readouts for evaluating potential therapeutic interventions

How might understanding BHLHE22 function contribute to regenerative medicine?

Given BHLHE22's role in neural development and differentiation, it has potential applications in regenerative medicine:

• Neural differentiation protocols:

  • Manipulate BHLHE22 expression to direct stem cell differentiation toward specific neuronal subtypes

  • Optimize temporal expression patterns to mimic developmental programs

  • Create reporter lines to track differentiation efficiency

• Organoid development:

  • Incorporate BHLHE22 modulation in brain organoid protocols to enhance cellular diversity

  • Use BHLHE22 expression as a marker for certain neuronal populations

  • Study the effects of BHLHE22 variants in patient-derived organoids

• Neural repair strategies:

  • Investigate whether BHLHE22 expression in transplanted neural stem cells improves integration into host neural circuits

  • Determine if modulating BHLHE22 in resident neural stem cells can enhance endogenous repair mechanisms

  • Study the role of BHLHE22 in axon regeneration after injury

• Disease modeling:

  • Use BHLHE22 variant-containing iPSCs to model neurodevelopmental disorders

  • Screen for compounds that rescue phenotypes in cellular models

  • Develop high-throughput assays based on BHLHE22 target gene expression

The deep understanding of BHLHE22's molecular function could significantly advance our ability to generate specific neuronal subtypes for cell replacement therapies and disease modeling.